Advances in imaging biological cells using optical tomography have been developed by Nelson as disclosed, for example, in U.S. Pat. No. 6,522,775, issued Feb. 18, 2003, and entitled “Apparatus and Method for Imaging Small Objects in a Flow Stream Using Optical Tomography,” the full disclosure of which is incorporated by reference. Further developments in the field are taught in Fauver et al., U.S. patent application Ser. No. 10/716,744, filed Nov. 18, 2003 and published as US Publication No. US-2004-0076319-A1 on Apr. 22, 2004, entitled “Method And Apparatus Of Shadowgram Formation For Optical Tomography,” and Fauver et al., U.S. patent application Ser. No. 11/532,648, filed Sep. 18, 2006, having a related application published on Mar. 27, 2008 as PCT Publication WO2008/036533, entitled “Focal Plane Tracking For Optical Microtomography,” the full disclosures of which are also incorporated by reference.
In the field of optical tomography continuous scanning from multiple perspectives is used to acquire projection images from, effectively, an infinite number of adjacent focal planes. In one example, the focal plane of an optical imaging system is mechanically translated along an axis perpendicular to the focal plane through the thickness of a specimen during a single detector exposure. This is often referred to as “scanning” the focal plane. The process is repeated from multiple perspectives, either in series using a single illumination/detection subsystem, or in parallel using several illumination/detection subsystems. In this way, a set of pseudo-projections is generated, which can be input to a 3D tomographic image reconstruction algorithm. The method disclosed may be useful in applications such as high resolution optical tomography of small objects.
Specimen preparation for optical tomography typically begins when patient specimens are received from a hospital or clinic. The specimens are processed to remove non-diagnostic elements, while retaining objects of interest, such as biological cells. Except in the case of live cells, specimens are fixed and then stained. Live cells may be stained, but are usually not fixed. Stained specimens are then mixed with an optical gel or fluid, inserted into a micro-capillary tube and images of objects, such as cells, in the specimen are produced using an optical tomography system. The resultant images comprise a set of extended depth of field images from differing perspectives called “pseudo-projection images.” The set of pseudo-projection images can be reconstructed using backprojection and filtering techniques to yield a 3-D reconstruction of a cell of interest.
The 3-D reconstruction then remains available for analysis, enabling the quantification and the determination of the location of structures, molecules or molecular probes of interest. An object such as a biological cell may be labeled with at least one stain or tagged molecular probe, and the measured amount and location of this probe may yield important information about the disease state of the cell, including, but not limited to, various cancers such as lung cancer, breast cancer, prostate cancer, cervical cancer and ovarian cancer.
Functional imaging of cells may be carried out using fluorescent optical projection tomography microscopy (FOPTM). Unfortunately, a common problem in fluorescence microscopy is photobleaching, in which some of the fluorophores permanently cease to emit light. This can occur over time periods ranging from seconds to minutes, and results in a reduction of the signal level, with a concomitant decrease on the signal-to-noise ratio.
Another problem that can occur during FOPTM is poor focus. Poor focus typically results from a focal-plane scan range that does not encompass the entire object thickness. This focusing error yields an image with lower contrast between dark and light regions than would otherwise be obtained.
Yet another problem may be introduced by the presence of a gradient in the light intensity across the field of view. A typical cause of this phenomenon is misalignment of the optical components in the camera system.
In the absence of these problems, the total light emission detected by an FOPTM camera is expected to stay the same for each of the pseudo-projections in a data set, since each pseudo-projection samples the entire volume and therefore all the fluorophores. There is an unmet need in the art to provide increasing amounts of accurate, detailed information about the cell structure at the functional and molecular level. This need is exacerbated by the fact that fluorescence presents a problem for conventional tomography, since fluorescence is typically produced by a light source within the reconstruction volume, along the projection path.
The present disclosure provides new and novel solutions to overcome problems due to photobleaching and other errors that may be present in an optical tomography microscopy system.